Functional disparity of distinct CD28 response elements toward mitogenic responses

Area of Science:

• Immunology
• Molecular Biology
• Cellular Biology

Background:

• T-cell activation requires antigen-specific T-cell receptor signaling and costimulatory signals.
• CD28 provides a major costimulatory signal for T cells, enhancing cytokine gene transcription.
• A CD28 response element (CD28RE) was previously identified in the interleukin-2 (IL-2) promoter.

Purpose of the Study:

• To investigate the function of the NF-kappaB sequence in the IL-6 promoter as a CD28 response element.
• To compare the binding specificities and functional capacities of different CD28 response elements.

Main Methods:

• Reporter gene assays to assess promoter activity.
• Electrophoretic mobility shift assays (EMSAs) to study protein-DNA interactions.
• Site-directed mutagenesis and domain-swapping experiments.

Main Results:

• The NF-kappaB sequence in the IL-6 promoter functions as a CD28 response element, mediating CD28-induced transcriptional activation.
• Mutating the IL-6 NF-kappaB sequence abolished CD28 responsiveness.
• The IL-6 NF-kappaB motif bound a broader range of NF-kappaB/Rel family proteins compared to the IL-2 CD28RE.
• Domain swapping demonstrated that IL-8 CD28RE and HIV-1 NF-kappaB motifs conferred IL-6 promoter responsiveness, unlike the IL-2 CD28RE.

Conclusions:

• Two distinct classes of CD28 response elements exist, differentiated by their NF-kappaB binding profiles.
• These differences in binding capacity dictate their ability to confer CD28 costimulatory signals to heterologous promoters.
• The IL-6 NF-kappaB motif represents a distinct class of CD28RE with unique regulatory properties.